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ABSTRACT Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the

rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic

acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4β2, but not α6 subunits. Polyamines are classically studied as regulators of ion

channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with

neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated

neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology. SIMILAR

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RESPONSE TO NICOTINE IN CULTURED SH-EP1 CELLS Article 12 July 2021 INTRODUCTION Neuronal nicotinic acetylcholine receptor (nAChR) ion channels mediate behavioral, cognitive and autonomic

effects of acetylcholine and addictive properties of nicotine1,2,3,4,5,6. The nAChR family comprises nine alpha (α2-10) and three beta (β2-4) subunits that combine to form an array of

pentameric cation channels throughout the brain and peripheral nervous systems. The major nACh receptors in brain are α7 homomers, α4β2 heteromers, and α6-containing heteromers. These

receptors are targets of numerous approved and experimental medicines for diverse conditions including Alzheimer’s disease, Parkinson’s disease, and neuropathic pain7,8. Whereas nAChRs are

compelling drug targets, progress is hampered because most receptor subtypes do not functionally express in the non-neuronal cell lines used for screening9,10. We previously identified

NACHO, a four-pass transmembrane protein that serves as a client-specific chaperone for assembly of most neuronal nAChRs11,12,13. In brain, NACHO is essential for function of α7 receptors,

and additional proteins, Ric-314 and certain Bcl-2 family members15, synergize with NACHO to enhance α7 assembly and surface trafficking. NACHO also promotes assembly of α4β2 receptors,

though Ric-3 and Bcl-2 proteins have minimal impact on α4β2. Here, we sought factors that conspire with NACHO to enhance function of α4β2 receptors, the most abundant nAChR subtype in brain.

These neuronal α4β2 receptors mediate diverse physiological actions of acetylcholine and underlie nicotine dependence16. Genome-wide cDNA screening for protein enhancers of α4β2 function

identified, quite surprisingly, a single standout clone that encodes spermidine/spermine acetyltransferase (SAT1), the rate limiting enzyme for polyamine catabolism. By degrading polyamines,

SAT1 promotes α4β2 function to an even greater extent than does NACHO. Polyamines are abundant polycations, including spermidine and spermine, that play multiple roles in cell growth,

differentiation and survival17. The interplay between their synthesis by ornithine decarboxylase-1 (ODC1) and their degradation by SAT1 controls polyamine levels. ODC1 is amongst the most

dynamically regulated of all human proteins, and ODC1 is a drug target in oncology and infectious disease17,18. SAT1 transcription is also highly-regulated, and its acetylation of polyamines

promotes their cellular export18. Interestingly, numerous large genomic studies link polymorphisms in SAT1 with suicidal behavior19. In neurons, polyamines play important roles in synaptic

transmission by conferring inward rectification to certain potassium channels, AMPA receptors and nACh receptors20,21,22. Polyamines also participate in the pathogenesis of neurodegenerative

disorders17 and the excitotoxicity associated with cerebral ischemia23. We now find that polyamines also control assembly of neuronal α4β2 and α7 receptors. By contrast, polyamines do not

modulate assembly of α6β4 nAChRs, AMPA receptors or any other ion channel tested. Whereas polyamines classically regulate channel gating by occluding the ion pore, polyamine regulation of

nAChR assembly instead relies on negatively charged residues within the α4 or α7 cytosolic loop. Neuropharmacology studies using wild-type and NACHO knockout mice show that lowering

polyamine levels selectively upregulates cerebrocortical α4β2 and α7 levels and enhances the neuroprotective properties of nicotine. These studies identify an unexpected role for polyamines

in controlling ion channel biogenesis and suggest new strategies in neuropharmacology. RESULTS GENOME-WIDE SCREENING IDENTIFIES SAT1 AS AN ENHANCER OF NACHR To identify novel regulators of

α4β2 receptors, we co-transfected human embryonic kidney 293T cells (HEK293T) with plasmids encoding α4 and β2 subunits along with individual constructs from a 5943-cDNA clone library from

the Broad Institute24. Transfected cells were stimulated with nicotine (100 µM) and intracellular Ca2+ was quantified with a fluorescence imaging plate reader (FLIPR) (Fig. 1a). As

previously shown11, α4β2 alone produced a small nicotine-evoked Ca2+ signal, and co-transfection with NACHO significantly enhanced this (Fig. 1a). High throughput screening identified a

single clone that profoundly augmented the nicotine-induced Ca2+ response—to levels much higher than NACHO; this clone encoded spermidine/spermine N1-acetyltransferase (SAT1) (Fig. 1a). We

evaluated the effect of SAT1 on other nAChRs and other Cys-loop receptors both by FLIPR and electrophysiology. Consistent with our screening results, SAT1 dramatically increased ACh-evoked

currents from α4β2 (Fig. 1c, d). SAT1 also synergized with NACHO to further enhance the α4β2-mediated Ca2+ influx (Fig. 1b, d), suggesting that SAT1 and NACHO employ different mechanisms.

Whereas SAT1 alone did not rescue homomeric α7 function, SAT1 powerfully synergized with NACHO to increase α7 mediated currents (Fig. 1b–d). In contrast, SAT1 had no significant effect on

α6β4 or 5-HT3A receptor function (Fig. 1b–d). SAT1, a small cytosolic protein, is the rate-limiting enzyme for polyamine catabolism (Supplementary Fig. 1). Together with polyamine oxidase,

SAT1 acetylates higher-order polyamines converting them to inactive forms that are transported out of cells18. By lowering polyamine levels, SAT1 effects on nAChRs could reflect

disinhibition, as polyamines can negatively affect nAChR gating22. This seemed unlikely for two reasons, First, α6-containing nAChRs are more sensitive to polyamine inhibition of gating than

are α725 whereas we find the opposite sensitivity to SAT1 in our functional assays (Fig. 1b–d). Second, polyamines do not block nAChRs at the hyperpolarized membrane potentials we used for

patch clamp studies22. As an alternative mechanism, we asked whether SAT1 increases nAChR surface expression. To assess this, we utilized extracellular HA-tagged subunits that allow

detection of surface receptors without disrupting channel function11. Strikingly, SAT1 boosted surface levels of both α4β2 and α7 (Fig. 2a, b). SAT1 also augmented surface trafficking atop

the effects of the α7 protein chaperones Ric-3, Bcl-XL, and Mcl-1, as well as the α7 chemical chaperone/orthosteric antagonist, methyllycaconitine (MLA) (Supplementary Fig. 2a, b) implying

that receptor upregulation by SAT1 involves a mechanism distinct from any previously described. SAT1 PROMOTES ASSEMBLY OF NACHRS BY CATALYZING POLYAMINES To determine whether effects on

nAChRs involve the catalytic activity of SAT1, we constructed a Tyr140Phe mutant that abolishes SAT1 enzyme activity26. This mutant SAT1 (mutSAT1) did not change surface expression of α4β2

or α7 receptors in the presence or absence of NACHO (Fig. 2a, b). Ornithine decarboxylase 1 (ODC1) is the rate-limiting enzyme for polyamine synthesis (Supplementary Fig. 1), and

difluoromethylornithine (DFMO) is an ODC1 active site inhibitor27 that depletes cellular polyamines18. Like SAT1 co-transfection, DFMO pretreatment augmented surface expression of α4β2 and

synergized with NACHO to further enhance α4β2 and α7 surface receptors (Fig. 2a, b). Pursuing the opposite tact, we found that co-transfection with the polyamine biosynthetic enzymes ODC1

and adenosylmethionine decarboxylase 1 (AMD1) diminished nAChR surface staining (Supplementary Fig. 3a, b). Neither catabolic (SAT1 co-transfection or DFMO pretreatment) nor anabolic (ODC1

and AMD1 co-transfection) polyamine manipulations affected surface expression of the 5-HT3A receptor (Fig. 2a, b; Supplementary Fig. 3a, b). We asked next whether polyamines influence nAChR

assembly, which can be probed with orthosteric ligands, such as [3H]epibatidine, that only bind at the interface between folded subunits28. Remarkably, either SAT1 or DFMO enhanced

[3H]epibatidine binding to α4β2 or α7 receptors (Fig. 2c). Furthermore, ODC1 and AMD1 reduced ligand binding to these receptors (Supplementary Fig. 3c). N1,N11-bis-(ethyl)-norspermine

(BenSpm) is a stable polyamine analog and SAT1 inhibitor26,29, and treatment of cells with 10 µM BenSpm specifically reversed the effects of SAT1 on nAChR surface expression (Fig. 2d). By

contrast, incubating cells with spermine, which does not penetrate cells and is rapidly degraded by SAT1, did not affect nAChR surface expression (Fig. 2d). Taken together, we find that

polyamines, independent of their established role in ion channel gating, enhance nAChR function by promoting surface expression and augmenting receptor assembly. POLYAMINES REGULATE NACHRS

ASSEMBLY VIA THEIR CYTOSOLIC LOOP Certain neuronal nAChRs display blunted ion flow at depolarized potentials. This inward rectification is mediated by polyamine binding to pore-lining

glutamate residues in the nAChR transmembrane domain 2 (TM2) corresponding to Glu247 in α422,30. It was previously shown that mutating this acidic residue to alanine (α4E247A) relieves

polyamine block of α4β2 channels in _Xenopus laevis_ oocytes22. Loss of negative charge in the α4E247A also reduces calcium permeability through the mutant receptor. Accordingly, we found

that a E247A α4 mutant (Fig. 3a) co-transfected with β2 evinced minimal nicotine-evoked Ca2+ influx in HEK293T cells (Fig. 3b), and this was unaffected by preincubation with DFMO or

co-transfection with SAT1. By contrast, DFMO or SAT1 enhanced surface expression of α4E247A/β2 similar to wildtype α4β2 (Fig. 3c, d). These data establish distinct mechanisms for polyamine

regulation of nAChR gating and trafficking. We next mutated Trp156 (Fig. 3e) in the α4 ligand-binding domain30. As expected, when co-transfected with β2, this α4W156A mutant was functionally

inactive and did not bind to [3H]epibatidine (Fig. 3f, g). By contrast, this mutant showed typical surface staining enhancement upon co-transfection with SAT1 cDNA (Fig. 3h, i) indicating

that polyamine regulation is independent of agonist binding. To identify nAChR regions responsible for regulation, we generated chimeras of α4 with α6, as surface expression of the latter is

not regulated by polyamines (Fig. 4a). We co-transfected these α4/α6 chimeras with β4 and quantified both receptor surface expression and nicotine-evoked Ca2+ signaling (Fig. 4). SAT1

co-transfection enhanced a chimera containing the α6 N-terminal and α4 C-terminal region but did not affect the converse chimera (Fig. 4a–c), which implies that polyamine regulation involves

the channel or the large cytosolic loop, which occurs between TM3 and TM4. We next constructed a pair of chimeras that swapped only this cytosolic loop. Quantification of receptor surface

expression and function showed that the α4 TM3-TM4 cytosolic loop is both necessary and sufficient for SAT1-dependent regulation of these nAChRs (Supplementary Fig. 4). Similarly, exchanging

the TM3-TM4 loop of homomeric α7 receptor with that of α6 (Supplementary Fig. 4d) abolished SAT1-mediated upregulation of alpha-bungarotoxin (α-Bgt) binding sites on α7 (Supplementary Fig. 

4e, f), which demonstrates that the intracellular loop determines polyamine-regulation of both α4 and α7 nAChR assembly. Previous studies noted that, within the T3-TM4 loop, a subdomain

proximal to TM4 contains conserved positively and negatively charged residues31. Interestingly, we observed certain negatively charged residues (Glu and Asp) in this subdomain that are

conserved between α7 and α4 but diverge in α6 to uncharged Asn (Supplementary Fig. 5a, b)31. Mutating those Glu and Asp to Ala in α7 (E438A or D451A) and α4 (E569A or D582A) blocked

SAT1-mediated upregulation of α7 assembly (Supplementary Fig. 5c, d) and α4β2 surface expression (Supplementary Fig. 5 e, f). By contrast, these mutations did not blunt effects of Ric-3 on

α7 or NACHO on α4β2. Replacing the corresponding Asn residues (N434 or N447) to Glu within the α6 cytosolic loop conferred moderate but significant SAT1 and DFMO sensitivity to α6β4

receptors (Supplementary Fig. 5g, h). These experiments identify negatively charged residues within the TM3-TM4 cytosolic loop that can mediate polyamine regulation of nAChR assembly and

surface expression. Future studies are needed to fully elucidate these mechanisms. POLYAMINES REGULATE NACHR GATING AND ASSEMBLY INDEPENDENTLY To further distinguish mechanisms for polyamine

control of nAChR assembly and gating, we compared effects of the membrane-permeant polyamine analog BenSpm with philanthotoxin-343 (PhTx-343), which cannot enter cells and engages the

channel pore via a long polyamine tail25,32. Neither BenSpm nor PhTx-343 preincubation changed basal surface levels of α4β2. Whereas BenSpm abrogated SAT1-mediated enhancement of surface

α4β2, PhTx-343 did not (Fig. 5a, b). By contrast, both BenSpm and PhTx-343 preincubation abolished nicotine-evoked Ca2+ with or without SAT1 co-transfection (Fig. 5c). These data demonstrate

that only cell-permeant polyamines can control of nAChR surface trafficking. To more completely elucidate these mechanisms, we compared effects of BenSpm and PhTx-343 on α4-containing and

α6-containing receptors, as SAT1 regulates trafficking of α4β4 but not α6β4 (Fig. 4b). As expected, pre-treatment with BenSpm, but not PhTx-343, reversed SAT1-mediated enhancement of α4β4

surface expression and neither had effect on α6β4 surface expression (Fig. 5d, e). By contrast, both BenSpm and PhTx-343 application abolished nicotine-evoked Ca2+ signaling from either α4β4

or α6β4 (Fig. 5f). We next evaluated effects of BenSpm and PhTx-343 on the α6/α4 chimeric constructs, which swap the extracellular N-terminal or TM3-TM4 cytosolic loop domains. As

predicted, the α4 cytosolic loop was necessary and sufficient for BenSpm pre-treatment to reverse SAT1-mediated enhancement of receptor surface expression, and PhTx-343 did not alter surface

expression for any chimera (Fig. 5e and Supplementary Fig. 6d, e). By contrast, acute application of either BenSpm or PhTx-343 blocked nicotine-evoked Ca2+-signaling from all receptor

chimeras in a concentration-dependent manner (Fig. 5f, Supplementary Fig. 6a–c). These molecular biological and pharmacological studies establish distinct mechanisms and protein domains for

polyamine control of nAChR trafficking and gating. POLYAMINES REGULATE ASSEMBLY OF NEURONAL NACHRS We next explored whether polyamines upregulate endogenous nAChRs in neurons. Accordingly,

we treated cultured rat cortical neurons with DFMO and quantified α7 nAChR surface expression with fluorescent α-Bgt. As a positive control, we incubated neurons with 100 μM nicotine33 and

found a significant increase in surface α-Bgt binding sites (Fig. 6a, b). Similarly, treating neurons with DFMO robustly increased surface α-Bgt binding (Fig. 6a, b). By contrast, neither

DFMO nor nicotine altered surface expression of the AMPA-type glutamate receptor subunit GluA1 (Fig. 6a, b). Preincubating neurons with DFMO also increased the nicotine-evoked Ca2+ signal

(Fig. 6c), an effect that was comparable to the enhancement seen following nicotine pretreatment. These nicotine-evoked Ca2+ responses reflect α7 activity, as they require the α7-specific

positive allosteric modulator (PAM) PNU-1205961534 and were abolished by the α7-specific inhibitor α-Bgt (Fig. 6c). [3H]Epibatidine binding in brain largely reflects α4β2 receptors and was

absent in cerebral cortex of α4 knockout mice35. Importantly, incubating cortical neurons with DFMO or transducing them with a lentivirus expressing SAT1 enhanced levels of [3H]epibatidine

binding (Fig. 6d), indicating an increased α4β2 receptor assembly. To confirm polyamine regulation of neuronal α4β2, we transduced cortical neurons with α4-expressing and β2-expressing

lentiviruses. Levels of [3H]epibatidine binding to the transduced neuronal membranes were ~20 times higher than untransduced neurons, and this was further increased with DFMO pretreatment

(Fig. 6d). Importantly, neither DFMO preincubation nor SAT1 transduction altered binding sites for the GABA receptor ligand [3H]flunitrazepam (Fig. 6d). Taken together, these data show that

polyamines regulate assembly of neuronal nAChR but not AMPA or GABA receptors. POLYAMINES MODULATE NICOTINE-MEDIATED NEUROPROTECTION Nicotine has well-established neuroprotective properties

and can mitigate excitotoxicity in vivo36 and in vitro37,38. This neuroprotection is mediated both by α739 and α4β2 nAChRs38,40. To assess the functional consequence of polyamine

upregulation of receptor assembly, we asked whether this nicotine-mediated neuroprotection could be enhanced by reducing cellular polyamine levels. We induced excitotoxicity by challenging

cultured neurons for one hour with glutamate (30 µM) and quantified cell survival and cytosolic cytochrome C (CytC), which is a trigger for apoptosis41,42. We also surface-labeled α7

receptors with fluorescent α-Bgt. In line with previous reports, glutamate challenge induced excitotoxity and increased cytosolic CytC (Supplementary Fig. 7a–c). Co-application of 100 μM

nicotine reduced excitotoxicity, but glutamate did not alter surface expression of α7 (Supplementary Fig. 7b, d). On the other hand, neurons pretreated with either DFMO or nicotine showed a

three-fold increase in surface α-Bgt-binding sites (Supplementary Fig. 7a, d) and a concordant reduction of glutamate-induced CytC release (Fig. 6e; Supplementary Fig. 7c). Nicotine or DFMO

preincubation also augmented nicotine-mediated protection from glutamate-mediated excitotoxic cell death (Fig. 6e and Supplementary Fig. 7b). To more directly link the neuroprotective

effects of DFMO with nAChRs, we studied NACHO KO mice, which have dramatically reduced nAChR function in brain12. We found that nicotine blunted glutamate-induced cell death in neurons from

wild-type but not NACHO KO mice (Fig. 7a–c). Fitting with results from our rat cortical neuron experiments, we found that pre-treating wild-type mouse neurons with DFMO increased surface

α-Bgt staining (Fig. 7e) and mitigated glutamate-induced increases in cytosolic CytC (Fig. 7d). By contrast, DFMO pre-treatment did not augment α-Bgt binding levels in NACHO KO neurons.

Accordingly, DFMO preincubation did not confer subsequent nicotine-induced protection from excitotoxic neuronal death in NACHO KO neurons (Fig. 7c). Collectively, these experiments show that

polyamines regulate functional assembly of nAChRs, which promotes neuroprotection in wild-type, but not NACHO KO neurons. DISCUSSION This study identifies an unexpected role for polyamines

in controlling assembly of neuronal α4β2 and α7 receptors. Our genome-wide analysis found that SAT1 increases assembly of α4β2 receptors to a much greater extent than any other protein

screened, including the nicotinic receptor chaperone NACHO. Polyamine-regulated assembly is specific for certain nAChRs, is distinct from other nAChR chaperone mechanisms, and requires

negatively charged amino acids within the α4 or α7 cytosolic loop. Blocking polyamine synthesis with DFMO upregulates neuronal α4β2 and α7 surface levels and promotes nicotine-mediated

neuroprotection, which provides a new angle for drug discovery. The biogenesis of pentameric nAChRs is a complex and tightly-regulated process43. Seminal studies in the 1980s found that

nicotine and other orthosteric ligands upregulate brain nACh protein levels, and this likely participates in nicotine dependence44,45. In _C. elegans_, Ric-3 is required for efficient

assembly of worm nAChRs46; in mammalian brain, NACHO is required for α711 and many other nAChRs12. Additional proteins synergize with NACHO for assembly of specific mammalian nAChRs. Ric-314

and certain Bcl-2 family proteins15 work with NACHO to promote function of α7, whereas BARP, LAMP5, and SULT2B1 conspire with NACHO to enhance function of α6-containing receptors13. Our

discovery of SAT1 regulation identifies the polyamine pathway as an additional controlling mechanism for α4β2 and α7 receptors. These multiple layers of nAChR regulation utilize distinct

mechanisms. In the case of α7, NACHO mediates subunit oligomerization11. Subsequent regulation by nicotine, Ric-3, and Bcl-2 proteins synergizes with NACHO to promote protein folding,

receptor surface expression, and channel function14,15. Nicotine and other orthosteric ligands promote assembly through α7’s ACh binding domain45 whereas Bcl-2 proteins15 and polyamines

engage the receptor cytosolic loop. However, polyamine regulation involves a distinct mechanism, as we find SAT1 augments receptor assembly atop NACHO, nicotinic ligand, Ric-3, or Bcl-2

family proteins. It is intriguing to ask why nAChRs—but not other receptors in the Cys-loop superfamily including 5-HT3 and GABAA receptors—require accessories for functional expression. One

possibility is that multiple assembly mechanisms provide regulatory nodes for controlling nAChR function. NACHO transcription is upregulated by physiological stimuli47, and Bcl-2 family

protein levels are dynamically-induced during developmental and pathological processes48. Furthermore, protein accessories may also determine nAChR cellular localization. The α6-containing

nAChRs specifically concentrate at presynaptic terminals of specific monoaminergic neurons, and this may be enabled by the lysosomal protein, LAMP5, which displays a similar restricted

distribution49. Polyamines are ubiquitous in biology and play multiple roles in cell growth, survival, and differentiation17. In neurons, polyamines control gating of several important ion

channels. Cytosolic polyamines confer inward rectification to certain potassium channels, AMPA receptors, and nAChRs20,21,22. Elegant biophysical studies showed that neuronal depolarization

draws cytosolic polyamines into the channel pore, which precludes ion flow20. This polyamine site is accessible to extracellular philanthotoxins50, which have long polyamine tails that can

engage the channel. Our studies decisively establish that regulation of α7 and α4β2 assembly by polyamines is distinct from their classical role in controlling ion channel gating. First, we

find that SAT1 increases α4β2 and α7 function even when recorded at hyperpolarized potentials that preclude nAChR gating control by cellular polyamines22 (Fig. 1c, d). Second, SAT1 promotes

assembly of non-functional α4β2 receptors that cannot bind ACh (Fig. 3e–i). Third, extracellular philanthotoxin acutely blocks α4β2 receptor function but philanthotoxin does not affect

receptor assembly (Fig. 5a–c). Fourth, chronic but not acute application of a cell-permeable polyamine analog, BenSpm, reverses the effects of SAT1 on α7 and α4β2 receptor assembly and

function (Fig. 2d). Interestingly, we find that SAT1 does not augment function of α6-containing nAChRs. Taking advantage of this α-subunit specificity, our α6/α4 chimeras determine that

polyamine regulation of channel assembly involves the α4 cytosolic loop. Again, this is distinct from the pore region that determines polyamine control of rectification. As α7 and α4β2 are

the most abundant nAChRs in human brain and control diverse aspects synaptic signaling and plasticity, polyamine regulation of their assembly has important physiological and

pathophysiological implications. Polyamine levels in neurons are dynamically regulated over both short-time and long-time scales. Acutely, synaptic transmission increases synthesis of

polyamines, which modulate integrative neuronal properties by reducing AMPA receptor currents51 and provide an excitability buffer by negatively regulating Na+ channels52. Long-lasting

changes in synaptic transmission associated with pathological processes such as epilepsy dramatically upregulate ODC153, which our study predicts would downregulate nAChR assembly. Indeed,

α4-containing nAChRs levels are decreased in piriform cortex of kindled mice54. Polyamines and nAChRs share compelling links to neuropsychiatric disorders. Numerous genetic, genomic, and

biochemical studies identify alterations in the polyamine pathway in major depressive disorder and suicide55. An SNP in the promoter region of SAT1 that reduces expression is associated with

suicide in a French-Canadian founder population55. Postmortem studies find reduced SAT1 protein and mRNA levels in precentral gyrus and cortical frontal lobe56. Also, polyamine levels

increase during anxiety episodes, and this polyamine stress response pathway19 could suppress nAChR assembly. Fitting with this, preclinical and early clinical studies suggest that α7 or

α4β2 agonists can improve depressive behavior in animal models and in depressed patients57. Numerous observations also link polyamine and nAChR alterations with cognitive and

neurodegenerative processes. Spermine synthase mutations cause Snyder-Robinson syndrome characterized by mental retardation and juvenile myoclonic epilepsy58, and these central symptoms are

also found in certain patients with mutations in α4 nAChR59. Interestingly, both SAT1 overexpression60 and nicotine36 protect animals from kainate-induced toxicity. Polyamine levels are

elevated17 and nAChR levels are reduced61 in Alzheimer’s disease, which is best treated today with cholinesterase inhibitors that augment nAChR activity. Furthermore, DFMO23 and nicotinic

agonists40 both mitigate neurotoxicity from neuronal ischemia. Our results that blocking ODC1 increases neuronal α4β2 and α7 and promotes nicotine-mediated neuroprotection provide a

mechanistic link for these observations. As such, targeting this polyamine/nAChR assembly pathway provides a new neuropharmacological strategy. METHODS GENES AND MOLECULAR BIOLOGY AND CELL

CULTURE The following genes are studied here: (Human forms) CHRNA4 (NM_000744.6), CHRNB2 (NM_000748.2), CHRNA7 (NM_000746.5), CHRNA6 (NM_004198), CHRNB4 (NM_000750), TMEM35A or NACHO

(NM_021637.2), Ric-3 (NM_024557.5), Bcl-XL (NM_138578.2), Mcl-1 (NM_021960.4), SAT1 (NM_002970.3), ODC1 (NM_002539); (mouse forms) 5HT-3A (NM_013561), AMD1 (NM_009665). Chimeric constructs

used in this study, with residues in the parenthesis are: α6NT/α4 (α61-239/α4243–628), α4NT/α6 (α41–242/α6240–495), α6/α4loop (α6328–465 exchanged to α4331–600), α4/α6loop (α4331–600

exchanged to α6328–465), α7/α6loop (α7328–457 exchanged to α6339–453). In brief, for α6NT/α4 chimera, α4243–628 region and α61–239 was linearized and amplified with PCR. The primers for

α61–239 contained complementary overhangs immediately upstream and downstream of amplified region. PCR products were ligated using In-Fusion HD Cloning Plus kit (Takara, 683910) in

accordance with manufacturer’s protocol. All other chimeras were generated following similar strategy and were confirmed by sequencing. Site-directed mutagenesis was done with two

complementary primer reactions (primer details provided in Supplementary Table 1), and all mutations were confirmed by sequencing. The α7-HA, β2-HA, and β4-HA constructs contained a PSGA

linker and HA tag immediately following the C-terminal residue. HEK293T cells (ATCC® CRL-3216™) were cultured in DMEM medium supplemented with 10% FBS and 1 mM sodium pyruvate. Cells were

seeded at 80-90% confluence and were transiently transfected using FuGENE®6 transfection reagents (Promega Corporation). All functional and expression assays in HEK293T cells were performed

after 48–72 h incubation at 37 °C following transfection, unless stated otherwise. All animal experiments reported here were overseen and approved by an AAALAC accredited institutional

review board. Primary cortical neurons were prepared from E18 rat cortex (supplied by BrainBits®) and E18 mice cortices from wild-type or NACHO knockout mice (TMEM35atm1(KOMP)Vlcg)11

(obtained from the laboratory’s vivarium). Cortices were dissociated using 10 U/mL papain (Worthington) digestion for 10 min, followed by trituration with a 10 mL glass pipette. Neurons were

seeded at 15,000 cells per well (384-well plates) and maintained in NbActiv4® media (BrainBits®). Immunostaining or FLIPR assays were performed at DIV 20 or DIV 13, respectively, following

6 days incubation in nicotine (100 µM) or DFMO (5 mM) as indicated. For radioligand binding assays, neurons were transduced with SAT1 or α4 and β2 encoding lentiviruses (DIV 7) or treated

with DFMO (DIV 14) and harvested on DIV 20. The lentiviral vectors encoding SAT1, α4 and β2 used a PGK promoter for transgene expression. The SAT1-expressing viral particles were packaged by

Vigene Biosciences™, while the α4-expressing and β2-expressing viruses were packaged by VectorBuilder. BROAD CDNA LIBRARY SCREEN The cDNA library used for α4β2 screening was from the Broad

Institute and contains 5943 cDNA clones. This genome-wide screen used a FLIPR assay on transfected HEK293T cells in 384-well plates. Each well was transfected with 60 ng of total cDNA with

the following composition: α4:β2:single Broad gene (2:4:3). Transfections with NACHO (α4:β2:NACHO [2:4:3]) served as positive control. FLIPR ASSAY FOR AGONIST-EVOKED CA2+ INFLUX

High-throughput FLIPR assays were conducted using 384-well BioCoat (Corning) plates. Cells were washed briefly in assay buffer (HyClone™ HEPES-buffered saline [GE Life Sciences] comprising

149 mM NaCl, 4 mM KCl, 10 mM HEPES, and 5 mM glucose at pH 7.4 and 300 mOsm osmolality, supplemented with 2 mM CaCl2 and 1 mM MgCl2) prior to Calcium5 dye (Molecular Devices) loading for 1 h

at RT. Following removal of excess dye, plates were placed in the FLIPR Tetra (Molecular Devices) chamber, and fluorescent Ca2+ signal was captured using ScreenWorks 4.0™ software

(Molecular Devices). To obtain neuronal α7 mediated FLIPR responses, we used 5 μM PNU-12059615—a selective drug that attenuates receptor desensitization. Tetrodotoxin, or TTX (500 nM) was

included in the assay buffer to inhibit spontaneous action potentials. IMMUNOFLUORESCENT STAINING AND IMAGE ANALYSIS HEK293T cells seeded on 384-well BioCoat plates (Corning) were incubated

with primary antibody against HA tag—Dylight 650® (Invitrogen™) in culture media for 1 h at 37 °C. Cells were fixed with 4% PFA (in HyClone™ HEPES-buffered saline) for 60 min, and after

washing, nuclei were stained with DAPI or NucBlue® reagent (Invitrogen™). For neurons, cells were fixed with 4% PFA for 1 h and then stained with fluorescent α-Bgt conjugates (1 μg/mL,

AlexaFluor 647, Invitrogen™, B35450) or primary antibodies for 1 h. Then cells were simultaneous permeabilized and blocked in 0.2% Triton X-100 and 10% normal goat serum for 30 min. To label

intracellular components, neurons were incubated in primary and secondary antibodies sequentially for 1 h each. Nuclei were stained with DAPI or NucBlue® reagent (Invitrogen™) prior to

imaging. Primary antibodies used were: HA (Mouse, 1:500, Invitrogen™ 2-2.2.14) conjugated to DyLight 650, GluA1 N-terminal (Mouse, 1:250, Millipore-Sigma RH95), Cytochrome C (Mouse, 1:100,

Invitrogen™ 7H8.2C12), MAP2 (Chicken, 1:1000, Millipore-Sigma AB15452). Secondary antibody used: goat anti-mouse IgG (H + L) AlexaFluor 488 (Invitrogen™, A-11001), goat anti-chicken IgY (H +

 L) AlexaFluor 555 (Invitrogen™, A-21437) at 1:1000 dilution. For quantification, images were acquired using Harmony™ high-content imaging software on an Opera Phenix™ screening instrument

(PerkinElmer) with a ×20 objective. Data were further analyzed with the Columbus™ data storage and analysis system (PerkinElmer). Viable cells were identified from the DAPI signal (area 

>30 μm2 for HEK293T; area >10 μm2 for neurons). Cell perimeter was determined from the nuclear staining, and area of the cytoplasm was calculated based on the cytoplasmic region of

interest. Average fluorescence intensity of wells was determined after subtracting the background signal, which was defined as anti-HA labeling of untransfected HEK293T cells or as α-Bgt647

labeling not displaced by 10 μM epibatidine. RADIOLIGAND BINDING ASSAYS HEK293T cells or cortical neurons were harvested in 50 mM ice-cold TrisHCl buffer (pH 7.4). Cells were homogenized for

30 s using the T-25 Ultra-Turrax homogenizer (Ika) and total protein concentration of the homogenate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Cell

homogenates were incubated with 10 nM [3H]epibatidine (for nAChR) or with 30 nM [3H]flunitrazepam (for GABA) in 96-well plates for 3 h at room temperature. Nonspecific binding was determined

by co-incubation of the cell samples with 10 μM unlabeled epibatidine or 100 μM unlabeled flunitrazepam. Assays were terminated by filtration through polyethylenimine-treated 96-well

Unifilter GF/B plates (PerkinElmer). Filter plates were washed with 500 mL TrisHCL buffer and then desiccated at 65 °C for 30 min. MicroScint-0 scintillant cocktail (50 μL; PerkinElmer) was

added to each well and plates read with a TopCount NXT scintillation counter (PerkinElmer). ELECTROPHYSIOLOGY HEK293T cells were seeded (1 million/well) on uncoated six-well plates and

transfected with cDNA combinations (total 2 μg/well) using FuGENE®6 transfection reagent (Promega Corporation). eGFP plasmid (10% of total cDNA) identified transfected cells. After 24 h,

cells were dissociated using CellStripper™ dissociation reagent (Corning) and re-seeded on 12 mm glass coverslips (40,000/well). Electrophysiological recordings were done 48 h after

transfection using external solution composed of HyClone™ HEPES-buffered saline (149 mM NaCl, 4 mM KCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2 and 5 mM Glucose at pH 7.4; 300 mOsm osmolality).

Intracellular solution contained (mM): 140 potassium gluconate, 10 HEPES, 4 Mg-ATP, 0.4 Na-GTP, and 0.6 EGTA (pH 7.3). To study most receptors, fast perfusion of compounds was achieved with

the Perfusion Fast-Step system (Warner Instruments). To study α7 nAChRs, ultra-fast perfusion of compounds was achieved with a piezo-driven perfusion system and theta glass (Siskiyou) on to

eGFP-expressing cells. The membrane holding potential was −70 mV. All recordings were performed at room temperature using an Axopatch 200B amplifier (Axon Instruments) and signals were

filtered at 2 kHz and digitized at 10 kHz. For α7 nAChRs, signals were filtered at 10 kHz with a digitization rate of 50 kHz. Data acquisition and subsequent analysis were done with pClamp9

software (Axon Instruments). STATISTICS Results are represented as mean ± SD unless stated otherwise. All FLIPR assay, immunostaining experiments and radioligand binding assay in HEK293T

cells and rat neurons were replicated thrice. Significance analyses between two datasets were performed with nonparametric Mann–Whitney _U_ test, while statistical analyses between three or

more datasets used one-way ANOVA (GraphPad Prism, Carlsbad, CA). Significance level α = 0.05 was set. REPORTING SUMMARY Further information on research design is available in the Nature

Research Reporting Summary linked to this article. DATA AVAILABILITY Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. A

reporting summary for this Article is available as a Supplementary Information file. The source data underlying Figs. 1b, d; 2b–d; 3b, c, g, i; 5b, c, e; 6b–e; 7c–e and Supplementary Figs. 

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Psychiatry_ 49, 200–210 (2001). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors are grateful to Wes Davini for his valuable inputs. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson and Johnson, 3210 Merryfield Row, San Diego, CA, 92121, USA Madhurima Dhara, Jose A. Matta, Min

Lei, Daniel Knowland, Hong Yu, Shenyan Gu & David S. Bredt Authors * Madhurima Dhara View author publications You can also search for this author inPubMed Google Scholar * Jose A. Matta

View author publications You can also search for this author inPubMed Google Scholar * Min Lei View author publications You can also search for this author inPubMed Google Scholar * Daniel

Knowland View author publications You can also search for this author inPubMed Google Scholar * Hong Yu View author publications You can also search for this author inPubMed Google Scholar *

Shenyan Gu View author publications You can also search for this author inPubMed Google Scholar * David S. Bredt View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS M.D. and M.L. performed cDNA library screening. J.A.M. conducted electrophysiology experiments. M.D. and H.Y. performed immunocytochemistry assays. M.D. conducted

radioligand binding assays. D.K. and S.G. provided chimeric cDNA constructs. M.D. and D.S.B. wrote the manuscript. All authors contributed to the discussion and editing of the paper. D.S.B.

supervised the project. CORRESPONDING AUTHOR Correspondence to David S. Bredt. ETHICS DECLARATIONS COMPETING INTERESTS All contributing authors are full-time employees in Johnson and

Johnson. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports

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assembly and implications for nicotinic acetylcholine receptor pharmacology. _Nat Commun_ 11, 2799 (2020). https://doi.org/10.1038/s41467-020-16629-3 Download citation * Received: 31 January

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